Journal of Advanced Research (2016) 7, 317–326

Cairo University

Journal of Advanced Research

ORIGINAL ARTICLE

Morphological, molecular and pathological

appraisal of Callitetrarhynchus gracilis plerocerci(Lacistorhynchidae) infecting Atlantic little tunny

(Euthynnus alletteratus) in Southeastern

Mediterranean

* Corresponding authors. Tel.: +20 2 1062368347, +20 2 35720399; fax: +20 2 35725240, +20 2 35710305.

E-mail addresses: m.abdelsalam@vet.cu.edu.eg (M. Abdelsalam), maawarda@eun.eg (M. Warda).

Peer review under responsibility of Cairo University.

Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.jare.2015.07.0042090-1232 � 2015 Production and hosting by Elsevier B.V. on behalf of Cairo University.

Mohamed Abdelsalam a,*, Rewaida Abdel-Gaber b, Mahmoud A. Mahmoud c,

Olfat A. Mahdy d, Nagwa I.M. Khafaga e, Mohamad Warda f,*

aDepartment of Fish Diseases and Management, Faculty of Veterinary Medicine, Cairo University, 11221, EgyptbZoology Department, Faculty of Science, Cairo University, EgyptcDepartment of Pathology, Faculty of Veterinary Medicine, Cairo University, EgyptdParasitology Department, Faculty of Veterinary Medicine, Cairo University, EgypteAnimal Health Research Institute, Agricultural Research Center – Dokki, Giza, EgyptfDepartment of Biochemistry, Biotechnology Center for Services and Researches, Faculty of Veterinary Medicine, CairoUniversity, Egypt

A R T I C L E I N F O

Article history:

Received 11 April 2015

Received in revised form 30 July 2015

Accepted 30 July 2015

Available online 8 August 2015

Keywords:

Euthynnus alletteratus

Mediterranean Sea fish

A B S T R A C T

The Atlantic little tunny, Euthynnus alletteratus, is widely distributed in temperate and tropical

waters of the Atlantic Ocean, Black and Mediterranean Seas. In this study, wild-caught little

tunny from Egypt, were found to be naturally infected with trypanorhyncha metacestodes,

and the overall prevalence rate of infection was 38.7%. The blastocysts were either loosely

attached to the mesentery of infected fish, or firmly attached and deeply embedded within the

hepatic parenchyma. These encysted plerocerci are identified as Callitetrarhynchus gracilis

(Trypanorhyncha, Lacistorhynchidae) based on its morphological and molecular characteriza-

tion. The morphological characteristics of C. gracilis including scolex shape; the bothridia

groove; the presence of frontal glands; the length of post-larval (appendix); metabasal armature;

318 M. Abdelsalam et al.

Trypanorhyncha

Callitetrarhynchus species

Histopathological studies

the existence of ‘Chainette’ and satellite hooks of different size were studied and described by

Light and Scanning electron microscope. The phylogenetic analysis of lsrDNA gene of

plerocerci confirmed the identification of the species to be deeply embedded in genus

Callitetrarhynchus. The histopathological examination revealed severe pathological changes in

the affected organs, including necrosis, inflammatory reactions, fibrosis and migratory tracts

of the parasitic larvae together with marked visceral organs adhesions. To the best of our

knowledge, this is the first report describing the detection of C. gracilis in little tunny collected

from the Abu Qir landing site in Alexandria, Egypt.

� 2015 Production and hosting by Elsevier B.V. on behalf of Cairo University.

Introduction

The Mediterranean Sea is considered as one of the main mar-ine biodiversity hotspots on the earth [1], and fish parasites area major component of such marine biodiversity [2]. Marine fish

is commonly infected with a high diversity of parasites thatcould be a potential threat to fish abundance [3], and larvalcestodes are some of the most damaging parasites to the vis-

cera of infected fish. Order Trypanorhyncha [4] is a cosmopoli-tan group of marine cestodes, with more than 270 recordedspecies [5]. They use three or four intermediate hosts in their

life cycles, before reaching the final host [6]. Larval try-panorhyncha encysted in visceral organs and musculature ofmarine teleosts, when being eaten by definitive hosts, and theyexcyst and form adult trypanorhyncha in the digestive tracts of

elasmobranchs; sharks and rays [7].The existence of larval trypanorhyncha in the fish flesh or

body cavity reduces the market value of the fish by making

them unappealing to consumers, thus causing economic losses[8]. Consumers may acquire this larval cestode through theconsumption of infected raw, undercooked, or inadequately

preserved fish [9]. There have been a few cases of accidentalhuman infections by trypanorhyncha and they may also causeallergic reactions [10,11].

The migration of plerocercoid larvae of trypanorhynchathroughout visceral organs is typically associated with hepaticnecrosis and extensive gonads and splenic damage [12]. Thismay reduce the reproductive capability and survival of affected

fish [12]. Heavy tapeworm infections result in a mechanicalobstruction of the gut and cause enteritis and degenerationof the intestinal wall [13]. Therefore, parasitological studies

on fish accompanied with histopathological response areimportant when encapsulated metacestodes are found in com-mercially important species.

Atlantic little tunny Euthynnus alletteratus [14] is a memberof family Scombridae that has wide distribution in theMediterranean Sea [15]. Based on the available data,Atlantic little tunny E. alletteratus is the most abundant spe-

cies among small tuna in Egypt and caught from theSoutheastern coast of Mediterranean Sea [16]. In spite of itseconomic important, E. alletteratus is still poorly studied

regarding its ichthyoparasitological problems.In Egypt, most of the previous studies were carried out on

trypanorhynchids from marine fish in the Red Sea [3,17,18],

and there are no records for their existence in little tunny fromthe Egyptian Mediterranean Sea.

Therefore, this study reported the infection of little tunny col-

lected from EgyptianMediterranean coasts with a species of thetrypanorhyncha cestode and provided information regarding its

histopathological effects on the host. Morphological investiga-

tions of the recovered parasite species were carried out by lightand scanning electron microscopy. In addition, the molecularanalysis was also conducted for accurate identification of this

parasite species.

Material and methods

Fish sample

During the period of October to December 2013, thirty-onespecimens of Atlantic little tunny; E. alletteratus were collectedby trap net method from the Coasts of Abu Qir landing site,Alexandria City, Egypt, located between longitude 29�47.10–29�50.40E and latitude 31�7.50–31�090N. The collected fish werepreserved in isothermal boxes supplied with ice and transferredto the laboratories of the Biotechnology Center for Services

and Researches, and fish diseases Department, Faculty ofVeterinary Medicine, Cairo University, where specimens wereidentified, measured, and submitted for necropsy. Fish was

medium-sized (19–28 cm long, and weighed 2025–3055 g).

Parasitic investigation

Fish samples were dissected for recovery of the prevailing par-asites. Body cavity and viscera were examined using a stereo-scopic dissecting microscope and the capsulated plerocerciwere removed from the infected organs. Walls of parasite blas-

tocysts were opened to remove the juvenile scoleces. The iso-lated worms were washed with saline solution and fixed in10% buffered formalin. The fixed specimens were stained with

acetic carmine, dehydrated and then mounted in Canada bal-sam and morphologically identified following the guidelinesof Carvajal and Rego [19] and Palm [10]. Drawing of speci-

mens was done by using the microscope tube (Nikon, Japan)Faculty of Veterinary Medicine, Cairo University, Egypt.Measurements were taken in millimeters.

Scanning electron microscope

For scanning electron microscopy, larvae were fixed in 4% glu-taraldehyde, washed in cacodylate buffer, dehydrated in

ascending alcohol series, processed in a critical point drier‘‘Bomer-900” with Freon 13 and sputter coated with gold–pal-ladium in a Technics Hummer V and then examined under an

Etec AutoScan at 20 kV JEOL scanning electron microscope(Etec, USA) in the Electron Microscope unit at Ain ShamsUniversity, Egypt. Measurements were taken in millimeters.

Fig. 1 Photomicrographs of the larval cestode

Callitetrarhynchus gracilis stained with acetic carmine and infect-

ing Euthynnus alletteratus. (a) Whole mounts of larval cestode

showing the bothridia (BO), followed by tentacles within tentacle

sheaths (TS) coiled till the bulb base (BU), four bulbs (BU), and

post-bulbosa area (PB). (b–g) High magnifications of: (b and c)

The anterior part is showing the four bothridia (BO), tentacles (T)

within tentacle sheaths (TS). (d) One of tentacles (T) within

tentacle sheaths (TS). (e) Four tentacle sheaths (TS) coiled till the

bulb base (BU). (f) The cephalic glands (frontal glands) do not

extend to the par bulbosa (BU). (g) Long Post-bulbosa area

(appendix) (PB).

Fig. 2 Drawing of stained C. gracilis. (a) The host capsule is

bladder-like to elongated. (b) Scolex showing distribution of

cephalic glands do not extend to the par bulbosa. (c) Posterior end

of appendix. (d–e) Metabasal tentacle armature showing external

face, note the chainette (The figure adopted from Mahdy et al. [17]).

Callitetrarhynchus gracilis plerocerci infecting Atlantic little tunny in Southeastern Mediterranean 319

Partial sequencing of lsrDNA and phylogenetic analyses

For molecular analysis, DNA from the preserved worm sam-ples was extracted according to the protocol of tissue GeneJet TM Genomic DNA purification Kit (Fermentas life

sciences, Lithuania). The D2 variable region (�600 bp) of thenuclear large subunit ribosomal DNA (lsrDNA) gene wassequenced to identify the plerocercoid. This region of thelsrDNA has been found to be informative for both diagnostic

and phylogenetic work in tetraphyllidean and related taxa[20,21]. Polymerase chain reaction (PCR) was carried out toamplify the target D2 variable region of lsrDNA using the fol-

lowing primers: 300F (5-CAA GTA CCG TGA GGG AAAGTT-3) and ECD2 (5-CTT GGT CCG TGT TTC AAGACG GG-3), as described by Aznar et al. [21], in a

25-ll reaction mixture comprising 1 ll of extracted genomicDNA, 5 ll of 1 mM deoxyribonucleotide triphosphates(dNTPs, MBI Fermentase), 0.25 ll of each primer

(50 pmol ll�1), 2.5 ll of 10� Taq polymerase buffer (MBIFermentase), 2 ll of 25 mM Mgc l, 1 ll Taq DNA polymerase

(2 U) (MBI Fermentase), and 13 ll of distilled water. The PCRcycle consisted of an initial denaturation step of 94 �C for4 min, followed by 40 cycles of 94 �C for 30 s, 52 �C for 30 s,

72 �C for 60 s, was finished with terminal extension at 72 �Cfor 7 min, and then rested at 4 �C. The PCR products were elec-trophoresed in 1.0% agarose gel in Tris–acetate–EDTA-

buffered gel stained with 1% ethidium bromide and visualizedwith a UV transilluminator. PCR products were purified usingstandard techniques (Qiaquick PCR Purification Kit, QiagenCompany, CA) and run against a standard mass ladder

(100 bp) on an agarose gel to estimate the concentration ofDNA. The PCR product was directly sequenced using theBigDye Terminator v3.1 Cycle Sequencing Kit (Applied

Biosystems, USA) with 310 Automated DNA Sequencer(Applied Biosystems, USA) using the same primers used inPCR. The sequence obtained was edited manually using

BioEdit version 7.0 [22], and aligned with other lsrDNAsequences available in GenBank using Mega 5 [23]. The phylo-genetic analysis was based on Kimura’s 2-parameter model forthe neighbor-joining method (substitutions included transver-

sions and transitions, pattern among lineages assumed

Fig. 3 Scanning electron micrographs of C. gracilis showing: (a) the whole body with 2 bothridia (BO), bulb base (BU) and parbulbosa

area (PB). (b–d) Scolex note the distinct bothridial groove near the border (BO). (d) Scolex with heart shaped bothridia, and armed

tentacle. (f and g). Tentacle with continuous spiral rows of hooks. (h and i) Metabasal armature showing external face, note the ’chainette’

and satellite hooks of different size.

320 M. Abdelsalam et al.

homogeneous, and the rate variation among sites uniform) with

1000 bootstrap replicates. The nucleotide sequences obtainedwere submitted to the GenBank under the accession numberKP300037.

Histopathological examination

Tissue specimens from liver and tissue masses showing adhesionbetween visceral organs and peritoneum were fixed in 10% neu-

tral buffered formalin for routine histopathological examina-tions. The fixed samples were washed in tap water overnightand exposed to ascend concentrations of ethanol (70%, 80%,

90% and 100%), cleared in xylene and embedded in paraffin.

Tissue slides of 5 lm thick sections were prepared and stained

with hematoxylin and eosin (H&E). The histopathologicalpreparation was performed according to Roberts [24].

Results

Clinical investigation

Naturally infected fish showed slight abdominal distension.The gross lesion revealed the presence of encysted try-panorhyncha larvae in mesentery, liver and other internal

organs within the peritoneal cavity. The encysted larvae wereslender or bladder-like in shape with white color. The older

Fig. 4 Dendrogram showing the relationship between the present C. gracilis with other Lacistorhynchoidea species recovered from

GenBank.

Callitetrarhynchus gracilis plerocerci infecting Atlantic little tunny in Southeastern Mediterranean 321

larval capsules, however, were brown to blue black and slightlyiridescent. The overall prevalence of 31 examined fish was38.7% (12/31).

Morphological description (based on 10 specimens)

The plerocercoid body was elongated and measured 9.1 ± 0.1

(8.7–11.5) mm in length and 1.1 ± 0.2 (1.0–1.5) mm in widthat the level of bulbs. The scolex supplied with four long cylin-drical and sheathed tentacles measuring 6.3 ± 0.2 (5.8–6.9)

mm in length (Figs. 1a–c and 3a–d). The scolex was measured6.9 ± 0.2 (5.4–6.3) mm in length with two short, heart-shapedbothridia (Fig. 3b–d). The bothridia were 1.1 ± 0.2 (0.91–1.4)

mm in length and 0.43 ± 0.2 (0.27–0.69) mm in width. It has aclear distinct bothridial groove near the border (Fig. 3d). Theanterior body has a frontal glands do not extend to the parbulbosa (Fig. 2a–b). Metabasal tentacular armature poeciloa-

canthus atypical external surface with chainette elements andintercalary hooks (Fig. 3f–g). A principal hooks from continu-ous half spiral rows of seven hooks beginning on the internal

surface; hooks 1 (10) their points are convergent and uncinate(supplement 2-B); hooks 2 (20) are uncinate; hooks 3–5 (30–50) are falciform (Fig. 3h–i); hooks 6–7 (60–70) are spiniform

and situated near external surface; principle hook 7 (70) andintercalary hooks a (a0) in satellite position to chainette ele-ments, the intercalary hook is smaller than the principle hook(7) (supplement 2-A). The tentacle sheaths were regularly

coiled until the base of the bulb and supplied with hooks incontinuous spirals (Fig. 1d and e). Four symmetricallyarranged bulbs were present at the end of a scolex. Each one

measured 1.1 ± 0.02 (0.87–1.05) mm in length and 0.29

± 0.02 (0.26–0.33) mm in width (Figs. 1a, f and 3a, b). Thepost-bulbosa (appendix) area was long with no granules, andmeasured 0.29 ± 0.02 (0.26–0.31) mm in length and 0.14

± 0.002 (0.11–0.20) mm in width (Fig. 1a and g).

Taxonomic summary

Parasites name: Callitetrarhynchus gracilis [25]

Family: Lacistorhynchidae [26]

Host: Little Tunny E. alletteratus [14] (Family: Scombridae)Locality: Coasts of Abu Qir landing site, Alexandria City,Egypt

Site of infection: Plerocerci larvae were found in the coelo-mic cavity of infected fishPrevalence of infection: 12 out of 31 examined fish were

infected (38.7%).Material deposition: Voucher specimens were deposited inthe Parasitology Laboratory, Zoology Department,Faculty of Science, Cairo University, Egypt.

Phylogenetic analysis

An approximately 560 bp fragment of the D2 variable regionof lsrDNA gene of the studied species was obtained.Comparison of the nucleotide sequences and divergence

showed that the present trypanorhynchid cestode is deeplyembedded in the genus Callitetrarhynchus, with 97% identitiesfor (FJ572957, AF286970, DQ642758) of C. gracilis, 96% for

(DQ642759) of C. speciosus, 95% for (AF286971) of Floricepsminacanthus, (DQ642761) of Lacistorhynchus dollfusi and

Fig. 5 (a) Histopathological section of parasitic nodules showing anterior part of the parasite with tentacle (T) and hooks (H). (b–d)

Histopathological section of fish viscera showing: (b) Parasitic larvae attached to intestinal serosa. Notice, the anterior part of the parasite

(A) in the peritoneal cavity (PC) near the intestinal and hepatic tissue (HT). (c) The anterior part of the parasite (P) in the abdominal

cavity surrounded with a thin layer of fibrous connective tissue (F), notice, the melanophores aggregation (M) in the hepatic tissue. (d)

Fish liver, with a cross section of parasitic larvae (CS) surrounded with a thin layer of fibrous connective tissue proliferation (F),

melanophores aggregation (M) and atrophied hepatocytes (AT), (H&E stain).

322 M. Abdelsalam et al.

(FJ572955) of Lacistorhynchus tenuis, 94% for (DQ642760) ofDiesingium lomentaceum, 93% for (DQ642765) of Grillotiarowei, (AF286967) of Grillotia erinaceus, and (DQ642763) of

Grillotia pristiophori. The present trypanorhynchid cestoderevealed sequence identities under family Lacistorhynchidae(P91%). The phylogenetic analysis revealed strong nodal sup-

port for two major lineages (Fig. 4). The first major clade rep-resents Lacistorhynchoidea species and consisted of two largersubclades, in which Pseudogilquiniidae, Mustelicolidae, and

Petrobothriidae are sister to Lacistorhynchoidae with weaknodal support. The other major clade stands for a mono-phyletic origin for Otobothrioidea species.

Pathological findings

The Gross examination of the affected fish showed the pres-ence of the parasites’ nodules in the abdominal cavity and it

evoked adhesion between the different visceral compartmentscausing difficulties in separating of individual organs. The par-asitic nodules were noticed only in the liver, intestinal serosa

and peritoneum causing adhesion of such parts.The histopathological examination revealed the presence of

multiple parasitic larvae attached to intestinal serosa. The

histopathological examination of the parasitic nodules revealed

the characteristic shape of the anterior part of the cestodal lar-vae in tissue section (Fig. 5a). A thin layer of fibrous connectivetissue surrounds the parasites and holds fast them to the intesti-

nal tissue (Fig. 5b). Remnants of the larvae were also noticed inthe hepatic tissue with prominent melanophores aggregation(Fig. 5c). Some cases reported characteristic passage tracts

formed of necrotic tissue with marked hepatocytes destruction.The parasitic larvae were wrapped with active thin layer of pro-liferative fibrous tissue, melanophores aggregation and atro-

phied hepatocytes (Fig. 5d). In such tracts, areas ofhemorrhage were frequently noticed along with melanophoresaggregates and mild to marked fibrosis (Fig. 6a).Mononuclear inflammatory cell infiltration (Fig. 6b) and hepa-

tocytes necrosis were common findings with multiple pyknoticnuclei in the affected hepatic tissue (Fig. 6c). The examinationof the spleen revealed marked activation of melano-

macrophage centers while the homeopathic tissue showeddepletion (Fig. 6d).

Discussion

This study reported the prevalence of infection with one of thetrypanorhyncha metacestodes in E. alletteratus which is a com-

mon pelagic species in Mediterranean fisheries. Larvae of

Fig. 6 (a) Histopathological section of fish liver showing migratory tracts (MT), melanophores aggregation (M), fibrosis (F) and

atrophied cells (AC) of hepatocytes. (b) Histopathological section of fish liver showing migratory tract of the parasite in hepatic tissue with

mononuclear inflammatory cells (IC) infiltration. (c) Histopathological section of fish liver showing necrosis of the hepatocytes (NH) and

pyknosis of the nuclei (PN). (d) Histopathological section of fish spleen showing the activation of melanomacrophage centers (MMC) and

depletion (D) of the hemopoietic tissue, (H&E stain).

Callitetrarhynchus gracilis plerocerci infecting Atlantic little tunny in Southeastern Mediterranean 323

trypanorhyncha were found to be encysted in mesentery, liverand other internal organs within the peritoneal cavity of E.alletteratus. Trypanorhyncha use crustaceans and invertebrateanimals as the first intermediate hosts [27,28], some of which

constitute food items for E. alletteratus, which act as the sec-ond intermediate host.

The recovered plerocercoid was identified as C. gracilis [25].

To our knowledge, the present finding of C. gracilis inAlexandria coasts represents its new geographical record inthe Southeastern Mediterranean Sea. This parasite was also

recorded from E. alletteratus in Turkey and the prevalence ratewas 91.3% [29], while it was 38.7% in this study. The presentfinding of C. gracilis in Egypt and Turkey indicates its

common occurrence in E. alletteratus of the MediterraneanSea. The existence of variation in C. gracilis prevalence inE. alletteratus from two different countries in theMediterranean Sea may indicate the presence of an uneven dis-

tribution in density of first intermediate hosts.C. gracilis was also isolated from more than 150 fish species

worldwide such as California [30], Brazil [27], Arabian Gulf

[28,31], and Red Sea in Egypt [3]. In Arab Gulf, Bates [32]and Abdou and Palm [3] recorded Callitetrarhynchus parasitefrom Scomberoides cammmersoniaus. The occurrence of

Callitetrarhynchus species in many species of teleosts suggesteda wide distribution of this parasite, and the existence of certainun-specificity of this parasite to its fish hosts.

Taxonomists are considered scolex shape, bothrial groove,

spread of cephalic glands, and the length of post-bulbosa.Tentacular armature was the most important characters for

trypanorhyncha taxonomy [19,33–36]. The morphologicalcharacters of C. gracilis found in the present study were similarto other Callitetrarhynchus species described previously. Suchsimilarity was represented by the presence of pars postbulbosa,

heterocanthus, homeomorphous, and unicate hooks that werearranged in continuous spirals. C. gracilis revealed specificcharacteristic morphological features that distinguish it from

C. speciosus. These morphological differences include thepresence of a clear distinct bothridial groove near the borderin C. gracilis, while it is weakly developed in C. speciosus;

the frontal glands do not extend to the par bulbosa inC. gracilis, while it extends to par bulbosa in C. speciosus;hooks 1 (10) in C. gracilis have their points convergent, while

in C. speciosus they are arranged in a parallel pattern; in C.gracilis the intercalary hook is smaller than the principle hookno. 7 (70), while in C. speciosus they are almost equal in size. Bymorphometrical comparison (Table 1), the parasite from the

present study mostly resembles to C. gracilis in Lethrinusnebulosus and in Carangoides malabaricusy [18,19,36,37], butshowing minor variation in the dimensions of the different

body parts.Palm et al. [7] accepted Otobothrioidea characterized by

bothrial pits as a superfamily, despite its derived placement

among the Lacistorhynchoidea. In our analyses, theLacistorhynchoidea grouped as sister to the Mustelicolidaespecies and thus we recognize both taxa as monophyleticsuperfamilies, which coincided with Olson et al’s study [38].

The present trypanorhynchoid showed the highest percentageof identity with other species within Lacistorhynchoidea. The

Table

1Comparativemeasurements

(inmillimeters)

ofthepresentCallitetrarhynchusgracilisandthose

described

previously.

Species

Host

fish

Dim

ensionsof

TotalbodyL

Pars

bothridialisL.

Pars

bulbosa

L.

Postbulbosa

L.

Callitetrarhynchusspeciosus[33]

Pomatomussaltatrix

13–17(15)

1.70–2.0

(1.92)

1.8–2.2

(2)

1.6–3.4

(2.0)

Callitetrarhynchusgracilis[34]

Lethrinusnebulosus

8.5–14.8

0.96–1.3

(1.13)

0.85–0.99(0.92)

(0.22–0.29)0.25

Callitetrarhynchusspeciosus[19]

Pagruspagrus

10.30–13.8

(12.97±

2)

0.8–1.2

(1.00±

0.2)

1.22–1.35(1.32±

0.02)

2.6–3.30(3.00±

0.2)

Callitetrarhynchusgracilis[35]

Carangoides

malabaricus

25

0.27

0.12

–

Callitetrarhynchusgracilis

(Thepresentstudy)

Euthynnusalletteratus

8.7–11.5

(9.1

±0.1)

0.91–1.4

(1.1

±0.2)

0.87–1.05(1.1

±0.02)

0.26–0.31(0.29±

0.02)

324 M. Abdelsalam et al.

phylogenetic analyses supported its taxonomic position withinthe genus of Callitetrarhynchus with indistinguishable relation-ship to other C. gracilis and C. speciosus. This finding was in

agreement with the previous reports of Olson et al. [38].Therefore, according to data from morphological andmolecular analyses, the present parasite belongs to family

Lacistorhynchoidea and classified as, C. gracilis with newlocality records in E. alletteratus from the Egyptian water.

Relatively few studies have investigated the effects of

trypanorhyncha on their hosts [39]. Paperna [40] reported thatencysted larvae of cestodes might not interfere with fish phys-iological functions and homeostasis, even when numerous inthe mesenteries. However, Adjei et al. [41] attributed an

increased mortality of Saurida tumbil due to the pressure ofC. gracilis blastocysts on the ventral aorta.

In this study, the gross examination of infected fish showed

severe adhesion in the internal viscera typically associated withthe presence of the encapsulated blastocysts. In some fish,adhesion with internal organs looks like a big mass of tissue.

This adhesion could be attributed mainly to the developmentand migration of plerocercoids within the host. In thisscenario, the parasitic cestodes with penetrative type scoleces

[42] elicited mechanical tissue damages that end up withchronic inflammatory lesions with adhesive nature. Herethe plerocercoids were noticed to be attached to the surfaceof the internal organs or frequently found loose in the

abdominal cavity. This finding agrees with the results ofAl-Niaeem et al. [39].

The observed plerocercoids were either migrating under the

wall of the intestine or dug inside the hepatic and splenic tis-sues causing their destruction. The inflamed sites in affectedtissues were recognized by aggregation of mononuclear cells

and melanophores. Such histological damage and inflamma-tory response were previously addressed by Bahram et al.[43]. Interestingly, no plerocercoids were found in the muscu-

lature of infected fish. These results revealed that these plero-cercoids mainly harbor the internal organs and, therefore, donot comprise the edible portion of the fish. Since the cestodeparasites (C. speciosus) were previously reported in muscula-

ture of two different edible fish species of Cephalopholishemistiktos and L. nebulosus, in the Arabian Gulf [44], furtherstudies should be required, however, to exclude the possibility

of musculature infestation in E. alletteratus fish species.There is still a considerable shortage in knowledge of tape-

worms from Scombridae fish, and considering existing infor-

mation about parasites from tunas, the number of speciesknown to parasitize these tunas is proportionally lesser thanthat of other fish, perhaps due to shortage of surveys of theirhelminth fauna.

Conclusions

This is the first record documents the infection of little tunny

fish by C. gracilis with 38.7% prevalence rate, and representsits new geographical record in Southeastern MediterraneanEgyptian coast. The infection was confirmed by both morpho-

logical and molecular tools. The infected fish showed encapsu-lated blastocysts-related visceral adhesion that attributed tothe mechanical damage induced by plerocercoids development

and migration.

Callitetrarhynchus gracilis plerocerci infecting Atlantic little tunny in Southeastern Mediterranean 325

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors wish to express their sincere gratitude to Dr. ShoShirakashi of the Kindai University (Japan) who did an excel-

lent review and supplied us with detailed notes and commentson this manuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jare.2015.

07.004.

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